The resurgence of tuberculosis (TB) coupled with the emergence of multi-drug resistant strains (MDR-TB) necessitates that novel therapies be developed. One strategy for the discovery of novel drugs is to identify the targets of effective agents. Pyrazinamide (PZA), one such agent, has a unique sterilizing activity. It is the inclusion of PZA with isoniazid and rifampin in current treatment regimens that constitutes the basis for 6-month short course therapy for M. tuberculosis (Mtb). PZA efficacy depends upon conversion of PZA to pyrazinoic acid (POA), the active agent. The lack of pyrazinamidase, an enzyme that converts PZA to POA, confers resistance to PZA. PZA is effective only against Mtb among the mycobacterial species, and then is effective only at an acidic pH. A recent study demonstrated that acidic pH enhances the intracellular accumulation of POA and suggested that M. tuberculosis, unlike other mycobactenal species, lacks an efficient POA efflux mechanism. Despite the clinical importance of pyrazinamide, there has been no integrated, comprehensive investigation of this compound. Although the mechanism of PZA activation and resistance is known, the precise mycobacterial function that is inhibited by POA remains unknown. Using 5-Cl-PZA as a selective agent for mycobacteria, we have found that 5-Cl-PZA and PZA inhibit fatty acid synthetase I FASI in Mtb. Preliminary studies have shown improved activity of PZA analogs against Mtb in infected human monocyte-derived macrophages and in murine models of infection. This integrative study proposes further elucidation of the mechanism of FASI inhibition to guide the generation of new analogs of PZA. We will test the activity of improved new compounds against PZA-resistant Mtb and against other species of mycobacteria and correlate this with FASI inhibition. We will compare the activity of new agents to PZA against intracellular bacilli (in macrophages), and assess the toxicity in human peripheral blood monocytes and cultured hepatocytes. Introduction of improved PZA analogs should result in more rapid sterilization of Mtb in a murine model of tuberculosis and therefore potentially lead to a shorter course therapy when tested in humans.